[Histonet] Immunocytofluorescence on smooth muscle cells

From:

"Bertrand Lefort"

Hello,
We are a new lab and we try to develop Immuno-cyto-fluorescence techniques
in the lab.
We are working with human bronchial smooth muscle cells.
I have currently a very big problem with all rabbit antibodies. All rabbit
antibodies (including IgG as isotype) give a non-specific signal, signal in
the nucleus and cytoplasm with very high intensity. There is no signal
between cells.
This problem does not exist with Rat and mouse antibodies I have used.
- I have tried different fixation methods (PFA 4%, acetone-methanol (1/1),
and kit like permeafix).
- I have tried different blocking solution (Rabbit serum 2%, FBS 2%, Horse
serum and Universal blocker solution from Dako) without any results.
- I have tried different diluents for my antibody (PBS 1X, PBS 1x-BSA 3%,
Dako diluents)
- I have tried different permeabilization methods (saponin, ptwx) (Not with
acetone-methanol fixation).
All I have tried gives always the same signal with a very high intensity
signal.
More details : I use a biotin-Goat-anti-rabbit for secondary antibody that
give not a signal if first antibody is omitted. I use PBS to dilute my
secondary antibody. I use Streptavidin-alexa to detect the signal.
I have also worked on endogen biotin on this cells and it does not appear to
be the problem.
Has someone reported this kind of problems on human smooth muscle cells ?
Has someone suggestions or solutions to this problem ?
Thanks in advance
Bertrand
Bertrand Lefort
Research Assistant
Laboratory of Neuro-immunology of Asthma
Research Center, Room J3155
5400, Blv. Gouin West
Montreal, QC
H4J 1C5
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